Transforming growth factor-beta (TGF-beta) belongs to a superfamily of structurally related regulatory proteins, which includes activin/inhibin and bone morphogenic proteins. Three isoforms of TGF-beta are produced in mammals: TGF-beta 1, TGF-beta 2, and TGF-beta 3. TGF-beta forms a homodimer, which typically is secreted as an inactive, latent complex, having two covalently linked propeptides (the latency-associated protein, or “LAP”), non-covalently bound to, and inactivating, the dimeric mature TGF-beta molecule. Activation of the latent complex in vivo occurs either through dissociation of the TGF-beta dimer from LAP (via proteases, nitric oxide, or other means), or through a conformational change in the latent complex caused by binding of LAP to either thrombospondin or αvβ6 integrin. Active TGF-beta dimmers specifically bind to TGF-beta Receptor II (TGF-beta RII), and typically bind two TGF-beta RII molecules. Binding by the TGF-beta homodimer recruits two TGF-beta Receptor I (TGF-beta RI) molecules, forming a heteromeric complex. Downstream signaling is mediated by the bound TGF-beta RI, a serine-threonine kinase, which is phosphorylated upon complexation to the TGF-beta/TGF-beta RII complex. Activation of TGF-beta RI causes phosphorylation of Smad2 and Smad3 and induces their heterodimerization with Smad4. The activated Smad complex then translocates to the nucleus where it regulates gene transcription.
TGF-beta regulates a plurality of processes, including cell differentiation and proliferation, migration, motility, deposition of the extracellular matrix, cell death, and immunosuppression. TGF-beta signaling can increase the synthesis of matrix proteins, such as vitronectin, fibronectin, laminin, tenascin, proteoglycans, and collagens, enhance the expression of cell adhesion molecules such as integrins, and increase the synthesis of various protease inhibitors. It also can decrease the synthesis of matrix degrading proteases.
TGF-beta, a potent immunosuppressant molecule, has profound inhibitory effects on several major immune system cell types, including T-cells (both CD4+ and CD8+), B lymphocytes, monocytes, macrophages, dendritic cells, polymorphonuclear leukocyte. Additionally, TGF-beta is a powerful chemoattractant for a plurality of types of immune cells, including T-cells (both CD4+ and CD8+), monocytes, PMNs, neutrophils and mast cells.
Different cells of the immune system have been characterized according to their activating (e.g., pro-inflammatory) or inhibitory (e.g., immunosuppressive) effects. Monocyte-lineage cells (e.g., macrophages) can be classified into the M1 (activating, inflammatory) and M2 (inhibitory) phenotype based on function and on expression patterns of proteins including cytokines, chemokines, surface receptors, apoptosis-related genes, soluble carriers, enzymes, extracellular mediators, and DNA binding factors. For example, certain proteins are expressed in these different monocyte cell types with greater than 200-fold differences. Differentiation of M1 versus M2 macrophages is well established and has been verified and extensively characterized based on different cytokine secretion profiles following E. coli lipopolysaccharide (LPS) challenge. After LPS challenge in culture, M1 macrophages overproduce tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-γ), whereas M2 macrophages overproduce monocyte chemotactic protein-1 (MCP-1) and interleukin-10 (IL-10).
Fc receptors are cell surface receptors, expressed on various immune cell types, that specifically bind Fc regions of immunoglobulin molecules. The Fc region is an effector-function conferring portion of the immunoglobulin constant region. Fc receptors are generally categorized according to the class of Ig molecule they recognize. For example, Fc gamma receptors (FcγR) bind Fc portions of IgG molecules. Mammalian Fc gamma receptors are further classified into four classes (FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16) and FcγRIV). The FcγRII class further includes the functionally distinct FcγRIIa and FcγRIIb sub-types. FcgRI has a high affinity for IgG Fc regions, can bind monomeric IgG at physiological concentrations of IgG, and has a restricted isotype specificity. FcγII and FcγIII receptors have low affinities for IgG Fc regions and typically only can bind multimeric IgG (for example, immune complexes and dimeric IgG) at physiological IgG concentrations.
Fc receptors, particularly Fc gamma receptors, can also be categorized into two functional classes: activating Fc receptors (including FcγRI, FcγRIIa, FcγRIII and FcγRIV) and inhibitory Fc receptors (including the sole naturally occurring inhibitory Fc receptor, FcγRIIb). Activating FcγRs are expressed on all myeloid cells, and their cross-linking results in sustained cellular responses. With the exception of T cells and NK cells, FcγRIIb is expressed in all cells of the immune system. When properly bound, FcγRIIb inhibits activation and proliferation of the cell in which it is expressed. Different cells of the immune system express different ratios of activating versus inhibitory FcγRs. For example, monocyte-lineage cells of the M1 (inflammatory) phenotype express overabundant amounts of activating FcγR, compared to the inhibitory FcγR. By contrast, M2-type monocyte-lineage cells display an overabundance in inhibitory FcγR expression compared to activating FcγRs (see Gordon, Nat. Rev. Immonol. 2005 December; 5(12):953-64).
Different FcγRs have varying affinities for particular subclasses of IgG molecules. For example, Nimmerjahn and Ravetch (Science, 310:1510-1512, 2005) reported that although all FcγRs can bind IgG immune complexes, individual Fcγ receptors display significantly different affinities for different IgG subclasses.
This report described the different affinities of particular IgG subclasses for functionally different Fcγ receptors as activating-to-inhibitory (A/I) ratios. The A/I ratio for each IgG subclass represented the affinity of the subclass for activating Fcγ receptors (either sFcγRIII or sFcγRIV) compared to its affinity for the inhibitory receptor (FcγRIIB).
The ratio, calculated based on affinities determined through in vitro binding to Fcγ receptors, accurately predicted the efficacy of particular IgG sub-classes to mediate immune function. For example, the ratio could predict the ability of specific isotypes of anti-tumor antibodies with identical CDRs (and thus, identical antigen specificity), to mediate immune effector responses. Differential efficiency of binding by various IgG subtypes to inhibitory and activating Fcγ receptors also has been described in humans (Armour, K L et al., Mol Immunol, 40:585-593, 2003; Bruhns, P. et al., Blood Nov. 18, 2008, Epub ahead of print).
Cancer is the second leading cause of death by disease in the United States, resulting in 1 of every 4 deaths. There is growing evidence that the microenvironments of tumors are overabundant in tumor-associated macrophages (TAMS) and myeloid-derived suppressor cells (MDSCs). These cells of myeloid origin and M2 phenotype, help to create an immunosuppressive environment that dampens anti-tumor immune responses.
Autoimmune diseases are responsible for a great deal of morbidity and mortality throughout the world, and have been described as the most prevalent group of diseases in the world. Autoimmune diseases are among the leading causes of death among young and middle aged women in the United States. Because these diseases tend to be chronic, they have a significant impact on medical utilization, direct and indirect economic costs, and quality of life. The prevalence of all autoimmune diseases has been estimated at over 5% of the US population, and most of its victims are women.
Rheumatoid arthritis (RA) is an inflammatory autoimmune disease caused by the attack by the immune system on the joints of an individual. RA is painful and often debilitating. RA affects women three times more often than men. RA affects more than 1% of the US population.
Current treatments include anti-inflammatory medication and anti-tumor necrosis factor-alpha therapies. Inflammatory macrophages (e.g., M1 phenotype macrophages) and B cells contribute to both inflammation and tissue destruction in RA.
Available proteins for treatment of cancer and autoimmune diseases are limited. For example, protein therapeutics with increased efficacy, half-life and specificity are needed. As an immunosuppressant and immune cell chemoattractant, TGF-beta is an attractive candidate for producing therapeutic effects on cells of the immune system. However, available TGF-beta proteins and their use in treating diseases are limited.
In particular, there is a need for TGF-beta proteins and complexes, including targeted TGF-beta proteins and complexes, with improved in vivo half-lives, and for TGF-beta proteins and complexes having specificity for particular cell types and specific effects on particular immune system and immune cell function. For example, TGF-beta proteins and complexes that do not have pleiotropic effects or have reduced pleiotropic effects are needed. Accordingly, it is among the objects of the invention to provide TGF-beta proteins, complexes (e.g., multimers, such as dimers), including proteins and complexes with high efficacy, specificity, availability and half-life, and combinations and compositions containing the TGF-beta proteins, and methods for treating diseases with the proteins and complexes.